The scaffold protein for iron-sulfur cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25°C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn (II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S → D reaction, and to decrease the rate of the D → S reaction. In the saturated IscU:IscS complex, IscU is largely disordered. In vitro cluster assembly reactions provided evidence for the functional importance of the S ⇆ D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed cluster assembly; variants that favor the D state were found to assemble less stable clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the cluster once it is assembled.amino acid sequence effects on protein stability | protein order-disorder transition | two-dimensional exchange spectroscopy | biogenesis of Fe-S clusters I ron-sulfur (Fe-S) clusters, which are among the most ancient and ubiquitous protein prosthetic groups, function in electron transport, enzymatic catalysis, and chemical sensing reactions or as structural units (1). Humans and other higher eukaryotes utilize the ISC (iron-sulfur cluster) system as the essential Fe-S cluster assembly mechanism in mitochondria, and defects in this system have been linked to a large number of human diseases (2). The prokaryotic ISC system has served as a useful model for understanding Fe-S cluster assembly and delivery. The bacterial system utilizes several proteins that have eukaryotic homologs: IscU (scaffold protein), IscS (cysteine desulfurase), HscB (cochaperone), HscA (chaperone), and CyaY (regulation or iron delivery, analog of human frataxin) (1). Interactions among these proteins have been shown to be critical for efficient Fe-S cluster biogenesis (3). The IscU protein acts as a scaffold on which the Fe-S clusters are assembled and from which the clusters are transferred to various apoproteins. IscS is a homodimeric pyridoxyl-5′-phosphatedependent cysteine desulfurase (4). Each IscS subunit binds an IscU molecule and transfers sulfane sulfur generated from the conversion of cysteine to alanine to the cluster ligand cysteines of IscU (5). HscA and HscB, the DnaK-like chaperone and the DnaJ-like cochaperone pro...

IscU is a scaffold protein that functions in iron-sulfur cluster assembly and transfer. Its critical importance has been recently underscored by the finding that a single intronic mutation in the human iscu gene is associated with a myopathy resulting from deficient succinate dehydrogenase and aconitase [Mochel, F., Knight, M. A., Tong, W. H., Hernandez, D., Ayyad, K., Taivassalo, T., Andersen, P. M., Singleton, A., Rouault, T. A., Fischbeck, K. H., and Haller, R. G. (2008) Am. J. Hum. Genet. 82, 652-660]. IscU functions through interactions with a chaperone protein HscA and a cochaperone protein HscB. To probe the molecular basis for these interactions, we have used NMR spectroscopy to investigate the solution structure of IscU from Escherichia coli and its interaction with HscB from the same organism. We found that wild-type apo-IscU in solution exists as two distinct conformations: one largely disordered and one largely ordered except for the metal binding residues. The two states interconvert on the millisecond time scale. The ordered conformation is stabilized by the addition of zinc or by the single-site IscU mutation, D39A. We used apo-IscU(D39A) as a surrogate for the folded state of wild-type IscU and assigned its NMR spectrum. These assignments made it possible to identify the region of IscU with the largest structural differences in the two conformational states. Subsequently, by following the NMR signals of apo-IscU(D39A) upon addition of HscB, we identified the most perturbed regions as the two N-terminal β-strands and the C-terminal R-helix. On the basis of these results and analysis of IscU sequences from multiple species, we have identified the surface region of IscU that interacts with HscB. We conclude that the IscU-HscB complex exists as two (or more) distinct states that interconvert at a rate much faster than the rate of dissociation of the complex and that HscB binds to and stabilizes the ordered state of apo-IscU.

IscU from Escherichia coli, the scaffold protein for iron-sulfur cluster biosynthesis and delivery, populates a complex energy landscape. IscU exists as two slowly interconverting species: one (S) is largely structured with all four peptidyl–prolyl bonds trans; the other (D) is partly disordered but contains an ordered domain that stabilizes two cis peptidyl–prolyl peptide bonds. At pH 8.0, the S-state is maximally populated at 25 °C, but its population decreases at higher or lower temperatures or at lower pH. The D-state binds preferentially to the cysteine desulfurase (IscS), which generates and transfers sulfur to IscU cysteine residues to form persulfides. The S-state is stabilized by Fe–S cluster binding and interacts preferentially with the DnaJ-type co-chaperone (HscB), which targets the holo-IscU:HscB complex to the DnaK-type chaperone (HscA) in its ATP-bound from. HscA is involved in delivery of Fe–S clusters to acceptor proteins by a mechanism dependent on ATP hydrolysis. Upon conversion of ATP to ADP, HscA binds the D-state of IscU ensuring release of the cluster and HscB. These findings have led to a more complete model for cluster biosynthesis and delivery.

Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S0 is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.

Background: IscU populates structured (S) and disordered (D) conformational states. In Fe-S cluster delivery, IscU interacts with HscB and HscA.Results: NMR reveals differential interactions of the S- and D-states with HscA and HscB.Conclusion: HscA and HscA-ADP bind preferentially to the D-state. HscB binds preferentially to the S-state. HscA-ATP binds neither the S- nor D-state tightly.Significance: Fe-S cluster transfer is coupled to chaperone-mediated disordering of IscU.

Protein misfolding and aggregation are pathological events that place a significant amount of stress on the maintenance of protein homeostasis (proteostasis). To prevent and repair protein misfolding and aggregation, cells are equipped with robust mechanisms that mainly rely on molecular chaperones. Two classes of molecular chaperones, heat shock protein 70 kDa (Hsp70) and Hsp40, recognize and bind to misfolded proteins, preventing their toxic biomolecular aggregation and enabling refolding or targeted degradation. Here, we review the current state of structural biology of Hsp70 and Hsp40-Hsp70 complexes and examine the link between their structures, dynamics, and functions. We highlight the power of nuclear magnetic resonance (NMR) spectroscopy to untangle complex relationships behind molecular chaperones and their mechanism(s) of action.

The highly-conserved protein, IscU, serves as the scaffold for iron-sulfur cluster assembly in the ISC system common to bacteria and eukaryotic mitochondria. The apo-form of IscU from Escherichia coli has been shown to populate two slowly interconverting conformational states: one structured (S) and one dynamically disordered (D). Furthermore, single-site amino acid substitutions have been shown to shift the equilibrium between the metamorphic states. Here, we report three-dimensional structural models derived from NMR spectroscopy for the S-state of wild-type (WT) apo-IscU, determined under conditions where the protein was 80% in the S-state and 20% in the D-state, and for the S-state of apo-IscU(D39A), determined under conditions where the protein was ~ 95% in the S-state. We have used these structures in interpreting the effects of single site amino acid substitutions that alter %S = (100×[S])/([S]+[D]). These include different residues at the same site, %S: D39V > D39L > D39A > D39G ≈ WT, and alanine substitutions at different sites, %S: N90A > S107A≈E111A > WT. Hydrophobic residues at residue 39 appear to stabilize the S-state by decreasing the flexibility of the loops that contain the conserved cysteine residues. The alanine substitutions at positions 90, 107, and 111, on the other hand stabilize the protein without affecting the loop dynamics. In general, the stability of the S-state correlates with the compactness and thermal stability of the variant.

The Escherichia coliisc operon encodes key proteins involved in the biosynthesis of iron–sulfur (Fe–S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the cysteine desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe–S cluster biogenesis. We have used a combination of NMR spectroscopy, small-angle X-ray scattering (SAXS), chemical cross-linking, and enzymatic assays to enlarge our understanding of the interactions of IscX with iron ions, IscU, and IscS. We used chemical shift perturbation to identify the binding interfaces of IscX and IscU in their complex. NMR studies showed that Fe2+ from added ferrous ammonium sulfate binds IscX much more avidly than does Fe3+ from added ferric ammonium citrate and that Fe2+ strengthens the interaction between IscX and IscU. We found that the addition of IscX to the IscU–IscS binary complex led to the formation of a ternary complex with reduced cysteine desulfurase activity, and we determined a low-resolution model for that complex from a combination of NMR and SAXS data. We postulate that the inhibition of cysteine desulfurase activity by IscX serves to reduce unproductive conversion of cysteine to alanine. By incorporating these new findings with results from prior studies, we propose a detailed mechanism for Fe–S cluster assembly in which IscX serves both as a donor of Fe2+ and as a regulator of cysteine desulfurase activity.